Although the sequencing of the first human genome was completed amidst much fanfare in 2003, a great need still exists for studying variability among different individual human genomes as well as among the genomes of other organisms. More than 90% of the genetic variability among humans is thought to consist of single-nucleotide polymorphisms (SNPs), and efforts are ongoing to map more than 300,000 SNPs.1, 2 While many SNPs have no significant impact on protein expression or cell function, specific SNPs have been found to predispose individuals to certain diseases, including sickle cell anemia and Alzheimer's disease.3, 4 For example, mutations in the p53 gene have been implicated in a wide variety of human cancers, with missense mutations comprising a large majority of deleterious p53 sequence alterations.5-9 Furthermore, sequence polymorphisms in a variety of interacting genes are suspected to be responsible for complex diseases such as cancer, heart disease and psychiatric disorders; the results of multiplexed, multigene SNP analyses in large populations are expected to enable valuable insights into such conditions.1, 10 
A wide variety of techniques has been proposed for SNP detection, and many of these methods have recently been reviewed.11, 12 Most methods begin with PCR amplification of the gene region to be tested, typically followed by an enzymatic allele discrimination reaction, and then the detection and identification of the reaction products. Biomolecule detection schemes based on fluorescence or fluorescence resonance energy transfer, mass spectrometry, or microarrays can allow accurate identification of allele-specific products. Each method has its advantages and disadvantages with respect to simplicity, sensitivity, ease of multiplexing, throughput, and cost; the choice of SNP genotyping method varies depending on the specific needs and resources of each laboratory.
One widely used technique for allele discrimination based on the synthesis activity of DNA polymerase is the single-base extension (SBE) assay, also known as mini-sequencing or primer-guided nucleotide incorporation.13-15 In this technique, an oligonucleotide primer is hybridized with its 3′ end immediately upstream of the locus to be genotyped. The SBE reaction is analogous to Sanger cycle sequencing,16 except that only chain terminators (ddNTPs) are included in the reaction. The DNA polymerase incorporates the ddNTP complementary to the target base on the template, and further extension of the DNA chain does not occur. A variety of different detection schemes can be used to determine the identity of the ddNTP that was incorporated and the genotype of the target allele. SBE followed by MALDI-TOF mass spectrometry,17, 18 solid-phase mediated fluorescence detection,19 or electrophoresis with 4-color LIF detection20 are all capable of multiplexed allele discrimination and detection.
Electrophoretic separation is an attractive method for separating SBE reaction products because capillary array electrophoresis (CAE) instruments are widely available; however, it tends to be a relatively costly approach to SNP detection,12 in part because CAE instruments are expensive to purchase and maintain. Increased throughput, either by higher order multiplexing (more SNPs per capillary) or shorter analysis time, is required to make electrophoretic separation competitive for SNP detection. Both of these goals can be achieved by using free-solution conjugate electrophoresis (FSCE) in place of conventional electrophoresis with a gel or polymer sieving matrix. FSCE, which is sometimes called end-labeled free-solution electrophoresis (ELFSE),21-24 is also expected to simplify the transition to microfluidic electrophoresis devices, which promise to be both faster and much less expensive than the bulky, complex CAE instruments, and to greatly expand the range of potential users of this technology.
What are needed are new tools and approaches for practicing FSCE so that its potential as a viable technology to aid in SNP detection is realized.